Tris(isonitrile)copper(I) adducts for preparing radionuclide complexes

ABSTRACT

Tris(isonitrile)copper(I) complex salts with anions selected from BF 4 , PF 6 , ClO 4 , I, Br, Cl and CF 3  COO are useful in preparing radionuclide complexes rapidly at room temperature. Preferred isonitrile ligands are ether isonitriles. The tris(isonitrile)copper(I) adducts enable technetium complexes, such as those of Tc99m, to be prepared easily just prior to their use as imaging agents.

BACKGROUND OF THE INVENTION

1. Field of Invention

This invention relates to tris(isonitrile)copper(I) adducts, theirpreparation, kits containing them, and methods of using them to preparetechnetium complexes.

2. General Background and Prior Art

Isonitrile complexes of various radionuclides and their use as imagingagents are known in the art as described, for example by Jones et al. inU.S. Pat. No. 4,452,774, issued June 5, 1984. The complexes described byJones, et al are of the general formula:

    [A((CN).sub.x R).sub.y B.sub.z B'.sub.z' ].sup.n

in which A is a radionuclide selected from radioactive isotopes of Tc,Ru, Co, Pt, Fe, Os, Ir, W, Re, Cr, Mo, Mn, Ni, Rh, Pd, Nb, and Ta, forexample, Tc99m, Tc99, ⁹⁷ Ru, ⁵¹ Cr, ⁵⁷ Co, ¹⁸⁸ Re and ¹⁹¹ Os; (CN)_(x) Ris a monodentate or polydentate isonitrile ligand bonded to theradionuclide through the carbon atom of the CN group; R is an organicradical; B and B' are independently selected from other ligands wellknown to those skilled in the art that result in isonitrile complexes,including solvents such as water, chloro and bromo groups, and ligandscomprising one or more neutral donor atoms capable of forming bonds withsaid radionuclide; x and y are each independently integers from 1 to 8;z and z' are each independently 0 or an integer from 1 to 7; with theproviso that (xy)+z+z' is less than or equal to 8; and n indicates thecharge of the complex and can be 0 (neutral), or a positive or negativeinteger the value of which depends upon the valence state of A, and thecharges on R, B and B'. Any desired counterion can be present asrequired by the charge on the complex with the proviso that suchcounterion must be pharmaceutically acceptable if the complex is to beused in vivo.

In the above formula, R is an organic radical that can be aliphatic oraromatic and may be substituted with a variety of groups which may ormay not be charged. When the organic radical R carries a chargedsubstituent group, the charge on the resultant complex is the summationof the charges of the ligands (R, B and B') and the charge of theradionuclide. Among the aromatic R groups which may be present arephenyl, tolyl, xylyl, naphthyl, diphenyl and substituted aromatic groupscontaining such substituents as halo, e.g., chloro, bromo, iodo orfluoro; hydroxy, nitro, alkyl, alkoxy, etc.; among the aliphatic Rgroups which may be present are alkyl, preferably containing 1 to 20carbon atoms, such as methyl, ethyl, n-propyl, isoprropyl, n-butyl,isobutyl, n-hexyl, 2-ethylhexyl, dodecyl, stearyl, etc. Substituentgroups may also be present in the aliphatic groups, including amongothers the same substituent groups as those listed above for aromaticgroups.

The complexes described by Jones et al. are described as being usefulfor visualizing cardiac tissue, detecting the presence of thrombi in thelung and associated areas of blood perfusion deficits, studying lungfunction, studying renal excretion, and imaging bone marrow and thehepatobiliary system.

In practice, the technetium complex of the simple hydrocarbonisonitriles such as t-butyliso-nitrile preferred by Jones et al. havedemonstrated somewhat high concentration in the lung and liver inhumans. [Holman, et al., J. Nucl. Med., 25, 1380(1984)].

Other isonitrile complexes of radionuclides are described by Jones etal. in European Patent Appln. 213,945 published Mar. 11, 1987. Theisonitrile ligands described therein have the formula:

    (CNX)R,

where X is a lower alkyl group having 1 to 4 carbon atoms, R is selectedfrom the group consisting of COOR₁ and CONR² R³ ; where R¹ can be H, apharmaceutically acceptable cation, or a substituted or unsubstitutedalkyl group having 1 to 4 carbon atoms, and R² and R³ can be the same ordifferent, results in a complex having the general advantages of theisonitrile radionuclide complexes of U.S. Pat. No. 4,452,774, but havinggenerally superior properties with respect to liver clearance or lungclearance. Consequently, these complexes can allow earlier imaging,and/or better imaging of bodily tissues and organs than theircorresponding parent compounds. Described are coordination complexes ofTc, Ru, Co, Pt or Re with the above isonitrile ligands.

Additional isonitrile complexes of radionuclides are described incoassigned U.S. patent application Ser. No. 056,003, filed June 1, 1987(NN-0181-B), in the names of Bergstein and Subramanyan. The isonitrileligands described therein are ether-substituted isonitriles of theformula: ##STR1## wherein A is a straight or branched chain alkyl group;and

R and R' each independently is a straight or branched chain alkyl groupor taken together are a straight or branched chain alkylene group,provided that:

(1) the total number of carbon atoms in A plus R in formula (I) is 4 to6, provided further that when the total number of carbon atoms is 6,then the carbon atom alpha to the isonitrile group is a quaternarycarbon, and

(2) the total number of carbon atoms in A plus R plus R' in formula (Ia)is 4 to 9.

The further evaluation of isonitrile Tc99m complexes of U.S. Pat. No.4,452,774 is described by E. Deutsch et al., J. Nucl. Med., 27, 409(1986); M. N. Khalil et al., Nucl. Med. Cummun., 6, 615 (1985); A. G.Jones et al., J. Nucl. Med., 25, 1350 (1984); A. G. Jones et al., Int.J. Nucl. Med. Biol., 11, 225 (1984); and A. Davison et al., Inorg.Chem., 22, 2798 (1983).

One difficulty in preparing isonitrile complexes of radionuclides isthat many isonitriles are extremely volatile; thus, the manufacturing oflyophilized kits for commercial purposes is not possible. PublishedEuropean Patent Appln. No. 211,424, published Feb. 25, 1987, addressesthis problem by preparing soluble isonitrile complexes of metals such asCu, Mo, Pd, Co, Ni, Cr, Ag and Rh and then reacting them with thedesired radionuclide. The pair of metals chosen is such that thenon-radioactive metal is readily displaceable from its isonitrilecomplex in an appropriate media by the desired radionuclide, thus givingthe desired radiopharmaceutical. The copper complexes described are(bis-isonitrile)phenanthroline and tetrakis-isonitrile complexes. Afurther difficulty occurs when such non-radioactive metal (e.g. Cu)isonitrile adducts are reacted with a desired radionuclide (e.g. Tc99m)to prepare a radiopharmaceutical. Many such adducts react with Tc99m atelevated temperatures to produce a radiopharmaceutical rapidly. Thereaction at room temperature is slow and may take several hours toproduce a high yield of the desired radiopharmaceutical.

SUMMARY OF THE INVENTION

According to the present invention, provided is atris(isonitrile)copper(I) adduct with an anion selected from BF₄, PF₆,ClO₄, I, Br, Cl and CF₃ COO. Such an adduct reacts easily and rapidlywith radionuclides such as Tc99m at room temperature to prepareradiopharmaceuticals in good yields.

Further provided is a kit for preparing a complex of an isonitrileligand and a radionuclide which comprises a predetermined quantity ofthe aforesaid copper tris adduct and a predetermined quantity of asterile, non-pyrogenic reducing agent capable of reducing apredetermined quantity of a predetermined radionuclide, preferablyTc99m.

Additionally provided is a process for preparing the aforesaid complexwhich comprises mixing in a suitable solvent the aforesaid copper trisadduct at room temperature with a radionuclide, preferablypertechnetate.

Also provided is a process for preparing the aforesaid adduct whichcomprises:

(1) reacting in a suitable solvent about one equivalent weight of:

(a) a tetrakis(acetonitrile)copper(I) BF₄, PF₆, or ClO₄, or

(b) cuprous iodide, bromide or chloride,

with about three equivalent weights respectively of:

(a) a tetrakis(isonitrile) ligand)copper(I) BF₄, PF₆, or ClO₄, or

(b) an isonitrile ligand; and

(2) removing the solvent to provide a solid copper tris product.

DETAILED DESCRIPTION OF THE INVENTION

The copper tris isonitrile adducts of the present invention can beprepared using any isonitrile ligand. Suitable isonitrile ligandsinclude those having, for example, the formula CNR where R is an organicradical which can be aliphatic or aromatic and can be substituted with avariety of groups which may or may not be charged. Examples of suitableisonitrile ligands can be found in the abovementioned U.S. Pat. No.4,452,774; Published European Pat. Appln. No. 213,945; and in U.S. Ser.No. 056,003, the disclosures of which are hereby incorporated byreference as described hereinbefore.

Tris(isonitrile)copper(I) tetrafluoroborates and perchlorates can besynthesized by the exchange of acetonitrile molecules intetrakis(acetonitrile)copper(I) complexes, [Cu(CH₃ CN)₄ X], where X isBF₄, PF₆, or ClO₄ (preferably BF₄ or ClO₄), with isonitrile ligandscontained in a complex of the formula [Cu(RNC)₄ ]X. Thus, when asuspension of one equivalent of [Cu(CH₃ CN)₄ ]X in a chlorinated solventsuch as chloroform is treated with three equivalents of [Cu(MIBI)₄ X]*,and the solvent is completely removed, a quantitative yield of[Cu(MIBI)₃ ]X is obtained. These compounds are white crystalline solidsat room temperature.

Tris(isonitrile)copper(I) iodides, bromides, or chlorides are preparedby reacting one equivalent of cuprous iodide, bromide or chloride withthree equivalents of an isonitrile, such as MIBI, in chloroform at about0° C. In this reaction, iodide is preferred.

The desired radiolabeled, isonitrile complexes are prepared by admixinga copper isonitrile adduct with a radioactive metal in suitable media attemperatures from room temperature to reflux temperatures or evenhigher. The radioactive metal (radionuclide) can be any of thosedescribed in U.S. Pat. No. 4,452,774 with technetium (Tc99m) beingpreferred. The desired labeled isonitrile complexes are isolable and canbe obtained in high yields. In some cases the isonitrile can itself actas a reducing agent thus eliminating the need for an additional reducingagent. Additional reducing agents, when required or desired to speed upthe reaction, are well known to those skilled in the art. Examples ofsuch well-known reducing agents include a stannous salt such as stannouschloride (often used with the isonitrile adduct in the form of a kit),formamidine sulfinic acid, sodium dithionite, sodium bisulfite,hydroxylamine, ascorbic acid, and the like. The reaction is generallycomplete after about 1 minute to about 2 hours, depending upon theparticular reagents employed and the conditions used.

In the case of technetium such as, for example Tc99 or Tc99m, anisonitrile complex is preferably made by mixing an appropriate reducingagent (capable of reducing technetium in aqueous medium) and anappropriate copper isonitrile adduct then adding pertechnetate.Alternatively, the copper isonitrile adduct and pertechnetate are mixed,then reductant added. For example, when [Cu(MIBI)₃ ]X, where X is BF₄,PF₆, ClO₄, iodide, bromide or chloride is reacted with ^(99m) TcO₄ ⁻ inaqueous media in the presence of a suitable reducing agent (e.g., SnCl₂/cysteine), high yields of [^(99m) Tc(MIBI)₆ ]⁺ are obtained. Thereaction takes place at room temperature and greater than 90% labelingis achieved within 5-45 min. The formation of [^(99m) Tc(MIBI)₆ ]⁺ isevidenced by radioanalytic thin layer chromatography (TLC) which showsgreater than 90% labeling within 30 minutes. The difference in thereactivities of tris and tetrakis complexes is readily apparent when onecompares the radioanalytical TLC of [^(99m) Tc(MIBI)₆ ]⁺ derived fromthese compounds at room temperature after twenty minutes reaction withpertechnetate.

An excess of the copper isonitrile adduct, up to 50-100 fold molarexcess or more, and an excess of reducing agent, can be used in thecomplexing reaction to ensure maximum yield from the technetium.Following the reaction, the desired complex can be separated from thereaction mixture, if required, for example by crystallization orprecipitation or by conventional chromatography or ion exchangechromatography; see U.S. Pat. No. 4,452,774, supra, the disclosure ofwhich is hereby incorporated by reference.

Kits in accord with the present invention comprise a sterile,non-pyrogenic, copper(I)tris adduct of an isonitrile ligand and an anionselected from BF₄, PF₆, ClO₄, I, Br and Cl, and, if required, a quantityof a reducing agent for reducing a preselected radionuclide. Preferably,such kits contain a predetermined quantity of the sterile copperisonitrile adduct and a predetermined quantity of a sterile reducingagent capable of reducing a predetermined quantity of the preselectedradionuclide. It is also preferred that the copper isonitrile adduct andreducing agent be lyophilized, when possible, to facilitate storagestability. If lyophilization is not practical, the kits can be storedfrozen. The copper isonitrile adduct and reducing agent are preferablycontained in sealed, non-pyrogenic, sterilized containers.

The invention can be further understood by the following examples inwhich parts and percentages are by weight unless otherwise indicated.

EXAMPLE 1 Preparation of [Cu(MIBI)₃ ]BF₄

A freshly prepared sample of [Cu(CH₃ CN)₄ ]BF₄ (0.533 g, 1.69 mmol) wassuspended in 20 ml of chloroform. To this was added 3.064 g (5.08 mmol)of [Cu(MIBI)₄ ]BF₄. The reaction mixture was stirred at room temperaturefor 15 min. to give a clear solution. The solvent was then evaporatedcompletely under reduced pressure to give a semi-solid residue which wasagain dissolved in 20 ml of chloroform. The solvent was once againevaporated under reduced pressure with the product beginning to solidifyunder high vacuum in about 2 hour. The crude product was then dissolvedin 25 ml of chloroform and to this 25 ml of ethylacetate was added. Theresulting clear solution was diluted with ethyl ether until it becamecloudy. It was warmed to about 30° C. and more ether was added to obtaina cloudy solution. The mixture was first allowed to cool to roomtemperature and was then chilled in a refrigerator overnight. Thecrystalline product was separated by filtration; (2.70 g, 81% yield),m.p. 112°-113° C.

EXAMPLE 2 Preparation of [Cu(MIBI)₃ ]ClO₄

A freshly prepared sample of [Cu(CH₃ CN)₄ ]ClO₄ (0.401 g, 1.226 mmol)was suspended in 20 ml of chloroform. To this was added 2.262 g (3.678mmol) of [Cu(MIBI)₄ ]ClO₄. After the reaction mixture was stirred for 15minutes to give a clear solution, the solvent was then evaporated todryness under reduced pressure. The residue was redissolved in 5 ml ofchloroform and then the solvent was once again evaporated completely.This process was repeated two more times to afford 2.411 g (98% yield)of white solid; m.p. 68°-69° C.

EXAMPLE 3 Preparation of [Cu(MIBI)₃ I]

To a stirred suspension of 3.040 g (16 mmol) of copper(I) iodide in 25ml of chloroform, under a nitrogen atmosphere, at 0° C. was slowly added5.424 g (48 mmol) of MIBI. The mixture was stirred until a clearsolution resulted and then was diluted with diethyl ether until thesolution became cloudy. The mixture was allowed to stand at roomtemperature overnight, after which the product was isolated byfiltration. It was washed twice with ether and dried under vacuum, 6.488g (77% yield), m.p. 85° C.

EXAMPLE 4

Technetium complexes were prepared by dissolving each of [Cu(MIBI)₃ I]and [Cu(MIBI)₃ ]BF₄ (1-2 mg), mannitol (15-25 mg), sodium citratedihydrate (2-3 mg), cysteine hydrochloride (1-2 mg) and stannouschloride (5-10 μL of a solution of 20-25 mg of SnCl₂.2H₂ O in 10 ml of0.01N HCl) in a 5 cc serum vial (pH 5-6). The vials were sealed and20-30 mCi of ^(99m) TcO₄ ⁻ obtained by elution of a 99Mo/99mTcradionuclide generator was added. The vials were allowed to sit for10-45 minutes at room temperature. The purity of the final product[Tc(MIBI)₆ ]⁺ thus obtained was determined by radioanalytic thin layerchromatography on Whatman C-18 reversed-phase plates using a solventmixture containing a 20% 0.5M aqueous ammonium acetate, 30% methanol,40% acetonitrile and 10% tetrahydrofuran.

As a control, the same technetium complex was prepared as above using[Cu(MIBI)₄ ]BF₄ as the starting adduct, except the serum vial was placedin a 100° C. water bath for 15 minutes after addition of thepertechnetate.

The complexes were evaluated by determining the biodistribution of eachin guinea pigs. Organ distribution of injected activity was determinedat 0.5, 15, and 60 minutes post-injection. For each time-point, threeguinea pigs were anesthetized with sodium pentabarbital (35 mg/kg ip)and injected with 0.1 ml of test material via the jugular vein. Theinjected dose of Tc99m isonitrile was 0.8-1 mCi. Upon sacrifice, theorgans were removed and radioactivity was measured using either aCapintec dose calibrator or gamma well counter. The heart, lungs andliver were weighed. The distribution of radioactivity in the heart,lung, and liver for the complexes are illustrated in Table I below. Itcan readily be seen that the technetium-99m complexes prepared fromtris(isonitrile)copper(I) adducts provide substantially the same imagingresults as the complex prepared from the tetra(isonitrile)copper(I)adduct.

                  TABLE I                                                         ______________________________________                                        Guinea Pig Biodistribution of Tc99m vs. Time                                  % ID/GR*                                                                      ______________________________________                                        Complex Prepared From [Cu(MIBI).sub.4 ]BF.sub.4                               TIME                                                                          POINT                                                                         (MIN)  0.5           15        60                                             ______________________________________                                        N      3             3         3                                              Blood  0.718 +/- 0.1706                                                                            0.03 +/-  0.009 +/- 0.0017                                                    0.0003                                                   Heart  1.967 +/- 0.4242                                                                            1.522 +/- 1.245 +/- 0.0791                                                    0.2716                                                   Lung   1.149 +/- 0.2934                                                                            0.879 +/- 0.374 +/- 0.1731                                                    0.3778                                                   Liver  0.579 +/- 0.0305                                                                            0.628 +/- 0.465 +/- 0.2326                                                    0.1892                                                   ______________________________________                                        Complex Prepared From [Cu(MIBI).sub.3 ]BF.sub.4                               TIME                                                                          POINT                                                                         (MIN)  0.5           15        60                                             ______________________________________                                        N      3             3         3                                              Blood  0.646 +/- 0.0697                                                                            0.020 +/- 0.014 +/- 0.0006                                                    0.0021                                                   Heart  1.898 +/- 0.3666                                                                            1.155 +/- 1.206 +/- 0.0879                                                    0.1102                                                   Lung   0.918 +/- 0.1472                                                                            0.414 +/- 0.335 +/- 0.0225                                                    0.0647                                                   Liver  0.888 +/- 0.1945                                                                            0.751 +/- 0.624 +/-  0.1217                                                   0.0662                                                   ______________________________________                                        Complex Prepared From [Cu(MIBI).sub.3 I]                                      TIME                                                                          POINT                                                                         (MIN)  0.5          15         60                                             ______________________________________                                        N      3            3          3                                              Blood  0.874 +/- 0.1341                                                                           0.034 +/-  0.020 +/- 0.0032                                                   0.0056                                                    Heart  1.548 +/- 0.6038                                                                           1.480 +/-  1.549 +/- 0.0970                                                   0.1866                                                    Lung   1.118 +/- 0.1827                                                                           0.450 +/-  0.445 +/- 0.1191                                                   .0982                                                     Liver  0.736 +/- 0.2603                                                                           0.732 +/-  0.569 +/-                                                          0.1235     0.1749                                         ______________________________________                                         *Percent injected dose per gram of tissue (mean +/- sample deviation).   

What is claimed is:
 1. A tris(isonitrile)copper(I) complex of theformula

    [Cu(R"NC).sub.3 ]X

wherein X is an anion selected from BF₄, PF₆, ClO₄, I, Br, Cl and CF₃COO; and R" is alkyl of 1-20 carbon atoms or has the formula: ##STR2##wherein A is a straight or branched chain alkylene group; andR and R'each independently is a straight or branched chain alkyl group or takentogether are a straight or branched chain alkylene group, providedthat:(1) the total number of carbon atoms in A plus R in formula (I) is4 to 6, provided further that when the total number of carbon atoms is6, then the carbon atom alpha to the isonitrile group is a quanternarycarbon, and (2) the total number of carbon atoms in A plus R plus R' informula (Ia) is 4 to
 9. 2. The adduct of claim 1 which istris(1-isocyano-2-methoxy-2-methylpropane)copper(I)BF₄.
 3. The adduct ofclaim 1 which istris(1-isocyano-2-methoxy-2-methylpropane)copper(I)ClO₄.
 4. The adductof claim 1 which istris(1-isocyano-2-methoxy-2-methylpropane)copper(I)I.
 5. A kit forpreparing a complex of an isonitrile ligand and a radionuclidecomprising a predetermined quantity of an adduct of claim 1 and apredetermined quantity of a sterile, non-pyrogenic reducing agentcapable of reducing a predetermined amount of a predeterminedradionuclide.
 6. A kit of claim 5 wherein said adduct and said reducingagent are lyophilized or frozen and said radionuclide is Tc99m.
 7. A kitof claim 5 wherein the reducing agent is SnCl₂ or a mixture of SnCl₂ andcysteine.
 8. A kit of claim 6 wherein said adduct istris(1-isocyano-2-methoxy-2-methylpropane)copper(I)BF₄.
 9. A kit ofclaim 6 wherein said adduct istris(1-isocyano-2-methoxy-2-methylpropane)copper(I)ClO₄.
 10. A kit ofclaim 6 wherein said adduct istris(1-isocyano-2-methoxy-2-methylpropane)copper(I)I.
 11. A kit of claim6 wherein the reducing agent is SnCl₂ or a mixture of SnCl₂ andcysteine.